The antibiotic linezolid targets the Jak2V617F mutation in polycythemia vera. Free

Current treatment options for polycythemia vera (PV) patients suppress excessive hematopoiesis, but lack specificity against JAK2^V617F+ cells. Our group initially aimed to study crosstalk between the microbiome and inflammation in PV, and unexpectedly identified Linezolid (LZD)–an FDA-approved antibiotic used to treat patients with gram-positive bacterial infections– as a JAK2^V617F selective inhibitor.

In JAK2^V617F mice, which recapitulate the PV phenotype (Li J., Blood, 2014), LZD treatment effectively normalized spleen sizes and disease associated hematopoietic parameters. Specifically, after 6 weeks of LZD treatment by way of drinking water (1g/L), hematocrit levels in JAK2^V617F mice were reduced from 77% to 51% (P=0.002), and hemoglobin, reticulocyte counts, and white blood cell counts were normalized to levels seen in healthy wild-type (WT) mice. Treatment of WT mice with LZD at 1 g/L for 6 weeks did not lead to any changes in hematological parameters, supporting the conclusion that LZD exerts a specific inhibitory effect on JAK2^V617F mice. Molecular docking studies revealed that LZD specifically interacted with the pseudokinase domain of JAK2^V617F protein but not with WT JAK2 protein–a key feature that distinguishes LZD from existing JAK2 inhibitors. In vitro treatment of homozygous JAK2^V617F+HEL cells with LZD suppressed cell proliferation, downregulated STAT5 signaling, altered cell cycle status, and increased apoptosis. By contrast, LZD did not inhibit cell proliferation of RS4:11 cells, which express WT JAK2, further confirming the specificity of LZD against JAK2^V617F. As a positive control, ruxolitinib–a JAK2 inhibitor that is currently widely used–significantly reduced cell proliferation of both HEL and RS4:11 cells, highlighting the unique selectivity profile of LZD.

PV patients contain healthy normal hematopoietic cells mixed with JAK2^V617F+ cells. To replicate this situation, we utilized a competitive bone marrow (BM) transplantation approach where BM cells from WT (CD45.1) and JAK2^V617F (CD45.2) mice were mixed and transplanted into lethally irradiated WT recipients. LZD treatment consistently rescued the PV-like phenotypes induced by JAK2^V617F+ cells with normalization of hematopoiesis and spleen size. Splenic fibrosis was significantly reduced in the LZD group compared to the untreated group (P=0.001). LZD significantly reduced the amount of JAK2^V617F mutant RBCs present in the peripheral blood circulation and consequently led to more WT RBCs in circulation (P=0.01), indicating a decrease in variant allele frequency(VAF). Most importantly, our data also demonstrated that LZD treatment in vivo lowered the JAK2^V617F+ hematopoietic stem cell (HSC) numbers and increased apoptosis of JAK2^V617F+ Megakaryocyte-Erythroid Progenitor (MEP) cells. LZD treatment of PV patient (N=6) mononuclear cells inhibited hematopoietic colony formation in a dose-dependent manner(10ug/ml, P=0.001; 50ug/ml, P<0.0001), with more primitive progenitor cells exhibiting greater sensitivity to this inhibition at the lowest dose tested (5ug/ml, P=0.002). This observation aligned with our animal data, which demonstrated that more primitive stem/progenitor cells were more susceptible to the effects of LZD. Additionally, LZD significantly reduced JAK2^V617F colony counts (P=0.03) with no change detected in WT colony counts(P=0.4), again confirming the specificity of LZD against JAK2^V617F mutant cells. Reduction of the JAK2^V617F–driven disease phenotypes across multiple model systems provided a firm foundation for the future evaluation of compounds structurally similar to LZD as a promising new therapeutic agent selectively targeting JAK2^V617F+ cells in PV patients.